In 1989, an endotoxin-induced serum activity that induced interferon-γ (IFN-γ) obtained from mouse spleen cells was described (Nakamura et al., 1989). This serum activity functioned not as a direct inducer of IFN-γ but rather as a co-stimulant together with IL-2, IFN-α/β, TNF or mitogens. An attempt to purify the activity from post-endotoxin mouse serum revealed an apparently homogeneous 50-55 kDa protein (Nakamura et al., 1993). Since other cytokines can act as co-stimulants for IFN-γ production, the failure of neutralizing antibodies to IL-1, IL-4, IL-5, IL-6, or TNF to neutralize the serum activity suggested it was a distinct factor. In 1995, the same scientists demonstrated that the endotoxin-induced co-stimulant for IFN-γ production was present in extracts of livers from mice preconditioned with P. acnes (Okamura at al., 1995). In this model, the hepatic macrophage population (Kupffer cells) expand and in these mice, a low dose of bacterial lipopolysaccharide (LPS), which in non-preconditioned mice is not lethal, becomes lethal. The factor, named IFN-γ-inducing factor (IGIF) and later designated interleukin-18 (IL-18), was purified to homogeneity from 1,200 grams of P. acnes-treated mouse livers. Degenerate oligonucleotides derived from amino acid sequences of purified IL-18 were used to clone a murine IL-18 cDNA (Okamura et al. 1995). Messenger RNAs for IL-18 and interleukin-12 (IL-12) are readily detected in activated macrophages. IL-18 does not induce IFN-γ by itself, but functions primarily as a co-stimulant with mitogens or IL-12. The human cDNA sequence for IL-18 was reported in 1996 (FIG. 1A SEQ ID NO:1).
Interleukin IL-18 shares structural features with the IL-1 family of proteins (Nakamura et al., 1993; Okamura et al., 1995; Ushio et al., 1996; Bazan et al., 1996). Unlike most other cytokines, which exhibit a four-helix bundle structure, IL-18 and IL-1β have an all β-pleated sheet structure (Tsutsui et al., 1996). Similarly to IL-1β, IL-18 is synthesised as a biologically inactive precursor (proIL-18), lacking a signal peptide (Ushio et al., 1996). The IL-1β and IL-18 precursors are cleaved by caspase 1 (IL-1β-converting enzyme, or ICE), which cleaves the precursors after an aspartic acid residue in the P1 position. The resulting mature cytokines are readily released from the cell (Ghayur et al., 1997 and Gu et al., 1997).
IL-18 is a co-stimulant for cytokine production (IFN-γ, IL-2 and granulocyte-macrophage colony stimulating factor) by T helper type I (Th1) cells (Kohnoet al., 1997) and also a co-stimulant for FAS ligand-mediated cytotoxicity of murine natural killer cell clones (Tsutsui et al., 1996).
Th1 lymphocytes are involved in the immune responses against tumors (Seki et al., 2000). Th1 responses include the secretion of the cytokines IL-2, 1L-12, IL-18 and IFN-γ, as well as the generation of specific cytotoxic T lymphocytes recognizing specific tumor antigens. The Th1 response is also a vital arm of host defence against many microorganisms. However, the Th1 response can also be associated with non-desirable effects such as the development of several autoimmune diseases, inflammation and organ transplant rejection.
Attempts to express the mature form of IL-18 in E. coli using a vector encoding the mature protein did not provide a fully active cytokine. An efficient expression system for the generation of the fully biologically active human IL-18 has been developed for therapeutic uses, e.g. in malignancies, or any condition where IFN-γ induction is desired (WO 00/61768). In this system, the IL-18 precursor caspase-1 cleavage site has been changed to a factor Xa site (ICE/Xa), and a vector encoding IL-18 ICE/Xa precursor was used for transformation of E. coli. Following expression of this IL-18 precursor in E. coli the mature IL-18 was generated by factor Xa cleavage in vitro. This mature IL-18 generated by factor Xa cleavage was fully active.
Cytokine binding proteins (soluble cytokine receptors) are usually the extracellular ligand binding domains of their respective cell surface cytokine receptors. They are produced either by alternative splicing or by proteolytic cleavage of the cell surface receptor. These soluble receptors have been described in the past, for example, the soluble receptors of IL-6 and IFN-γ (Novick et al., 1989), TNF (Engelmann et al., 1989; Engelmann et al., 1990), IL-1 and IL-4 (Maliszewski et al., 1990), IFN-α/β (Novick et al., 1994; Novick et al. 1992). One cytokine-binding protein, named osteoprotegerin (OPG, also known as osteoclast inhibitory factor-OCIF), a member of the TNFR/Fas family, appears to be the first example of a soluble receptor that exists only as a secreted protein (Anderson et al., 1997; Simonet et al., 1997; Yasuda et al., 1998).
An IL-18 binding protein (IL-18BP) was affinity purified, on an IL-18 column, from urine ( Novick et al., 1999). IL-18BP abolishes IL-18 induction of IFN-γ and of IL-8, activation of NF-KB in vitro and induction of IFN-γ in vivo. IL-18BP is a soluble circulating protein which is constitutively expressed in the spleen, and belongs to the immunoglobulin superfamily. The most abundant IL-18BP isoform, the spliced variant isoform a, exhibits a high affinity for IL-18 with a rapid on-rate and a slow off-rate, and a dissociation constant (Kd) of approximately 400 pM (Kim et al., 1999).
The residues involved in the interaction of IL-18 with IL-18BP have been described through the use of computer modelling (Kim et al., 1999) and based on the interaction of IL-1β with the IL1R type I (Vigers et al., 1997). In the model for IL-18 binding to the IL-18BP, the Glu residue at position 42 the and Lys residue at position 89 of IL-18 have been proposed to bind to Lys-130 and Glu-114 in IL-18BP, respectively (Kim et al., 1999).
IL-18 is constitutively present in many cells (Puren et al., 1999) and circulates in healthy humans (Urushihara et al., 2000). The high affinity of IL-1BP to IL-18 as well as the high concentration of IL-18BP found in the circulation (20 fold molar excess over IL-18), represents a unique situation in cytokine biology. Therefore, most, if not all, of the IL-18 molecules in the circulation is bound to the IL-18BP. The circulating IL-18BP which competes with cell surface receptors for IL-18, may act as a natural anti-inflammatory and an immunosuppressive molecule.
Viral agents encode IL-18BP like proteins, for example, M contagiosum viral proteins MC53 and MC54 share a significant homology to mammalian IL-18BP (Novick et al. 1999). M contagiosum proteins MC53 and MC54 possess the ability to bind and neutralize human IL-18 in a fashion similar to that of IL-18B (Xiang and Moss, 1999). The ectromelia poxvirus p13 protein, which is homologous to IL-18BP, binds human IL-18 and inhibits its activity in vitro. Mice infected with a p13 deletion mutant virus exhibited decreased levels of infectivity (Born et al., 2000). Therefore infectivity degree seems to correlate with the presence of IL-18BP.
The high levels of circulating IL-18BP may represent a natural defence against a runaway Th1 response to infection and development of autoimmune diseases. However, IL-18 contributes to the Th1 response which is important in host defence against tumors. Therefore, IL-18BP may bring about failure of the host to develop cytotoxic T cells directed against tumor cells. Indeed, there is evidence that IL-18 promotes host defence against tumors in mice. For example, in syngeneic mice, murine mammary carcinoma cells expressing murine IL-12 or murine IL-I8 were less tumorigenic and formed tumors more slowly than did control non-expressing cells (Coughlin et al., 1998). Antibody neutralisation studies revealed that the antitumor effects required IFN-γ. In another study, systemically administration of IL-18 to experimental animals in combination with B16 melanoma expressing B7-1 (CD80) resulted in dramatic suppression of melanoma formation, tumor growth, and a significant improvement in survival (Cho et al., 2000).
Cytokines are used as adjuvant to increase the effectivity of immunotherapy in cancer. For example, IL-2 is administered for renal cell carcinoma or melanoma (Gollob et al., 2000). Often, one important consequence of the treatment with cytokines is severe systemic toxicity profiles. Using cytokines, expressed by the patient's own tumor or dendritic cells, is a logical solution to the problem. Yet, if IL-18 will to be used locally, as adjuvant in tumor immunotherapy, the ability of the constitutive levels of IL-18BP to neutralize IL-18 in the local environment would still be exerted and consequently its effectivity is greately diminished.
The use of non-myeloablative allogeneic transplants, the so-called mini transplants, to treat leukaemia and solid tumors is increasingly successful in inducing graft-versus-leukaemia and graft-versus-tumor reactions (Slavin S., 2000; Slavin et al., 2000). Two studies that used either allogeneic peripheral blood stem cells (Childs et al. 2000) or dendritic cells (Kugler et al., 2000) to treat patients with metastatic renal cell carcinoma met a remarkable success. Although these studies need to be extended and confirmed, the concept that an ongoing graft-versus-tumor reaction is exploitable for immunotherapy in cancer is gaining acceptance (Slavin, 2000). Since IL-18 appears to be involved in these successful therapeutic approaches, a further improvement may be achieved if the neutralizing effect of IL-18BP can be abolished.
Mutants of IL-18 (IFN-γ, inducing factor) are described in EP0845530. The described IL-18 mutants are molecules in which 1, 2, 3 or all 4 cysteine residues in IL-18 (FIG. 1B) were replaced by serine or alanine residues. These mutants contained an intact consensus sequence (FIG. 1B). All the isolated mutants exhibit higher stability than wild type IL-18. The degree of stability of the mutants is directly proportional to the number of Cys residues replaced in the molecule. EP0845530 is silent on the ability of IL-18BP to neutralize these mutants.
The generation and therapeutic use of fully active IL-18 mutants unable to bind or bind with low affinity to IL-18BP, is therefore highly advantageous.